The vast majority of promoters in plants are unidirectional, with one upstream (5xe2x80x2) promoter directing only one gene which is 3xe2x80x2 to the promoter. It is often desirable to introduce multiple genes into plants for metabolic engineering, trait stacking and other purposes. It is also desirable to minimize repeated use of a single promoter, because repeated use may result in gene silencing. The present invention provides a strategy to make polar promoters bidirectional so that one promoter can direct the expression of two genes.
Common promoters are polar; that is, only one gene operably linked to a promoter can be expressed. In the present invention, in contrast, the inventors have found that a naturally-occurring unidirectional promoter can be bidirectionalized by fusing a minimal promoter at its 5xe2x80x2 end in an opposite orientation to the promoter (FIGS. 2 and 3), and that the engineered promoter can simultaneously direct the expression of two genes, with one gene on each end.
Gene expression consists of two major consecutive processes called transcription (from DNA to mRNA) and translation (from mRNA to protein/peptide). Transcription is often directed by a specific DNA sequence called a promoter that is in the upstream region of the transcribed DNA or gene. An eukaryotic promoter consists of two parts: one is called the core or minimal promoter, and the other comprises regulatory sequences or cis regulatory elements (FIG. 1). A minimal promoter is the DNA sequence that is immediately in the upstream of the transcription start site, and it generally contains a short sequence called the TATA box (FIG. 1). The role of a minimal promoter or TATA box is to provide a site for assembling the transcription initiation complex consisting of RNA polymerase II, transcription factor II B (TFIIB), TFIID, TFIIE, and TFIIH. However, the minimal promoter itself has no transcriptional activity. Its activation requires the cis regulatory elements on which other specific transcription factor(s) bind and subsequently interact with the transcription initiation complex to activate gene transcription. The arrangement of cis regulatory elements-minimal promoter determines that a promoter must be polar or unidirectional. Almost all eukaryotic promoters are unidirectional with one promoter directing one gene expression.
An array of promoters have been cloned and widely used for basic research and biotechnological applications. However, it is often needed to introduce multiple genes into plants for metabolic engineering and other purposes. To minimize or avoid repeated use of a single promoter (therefore minimize the potential for gene silencing), several strategies have been developed, including sequential transformation using multiple constructs with different selectable markers, co-transformation with multiple constructs, genetic crosses between plants transformed with different constructs, and fusion of multiple peptides with a plant virus proteinase-based protein splicing mechanism. The present inventors have invented a method as described below by which one can make one polar promoter bidirectional so that one promoter can be used to direct the expression of two genes, or gene fusions.
A typical eukaryotic promoter consists of a minimal promoter and other upstream cis elements. Lewin, B. Gene VI (Oxford University Press, Oxford, 1997), Odell, J. T., Nagy, F. and Chua, N.-H. Nature 313, 810-812 (1990), and Benfey, P. N. and Chua, N.-H. Science 250, 959-966 (1990). The minimal promoter is essentially a TATA box region where RNA polymerase II binds to initiate transcription, but itself has no transcriptional activity Benfey, P. N. and Chua, N.-H. Science 250, 959-966 (1990). The cis elements, upon binding by specific transcriptional factors, individually or in combination, determine the spatio-temporal expression pattern of a promoter. Benfey, P. N. and Chua, N.-H. Science 250, 959-966 (1990).
Artificial constructs as well as exception to nature""s rule have been disclosed previous to the present invention. None have described a bidirectional promoter as is described herein. Representative patents are summarized below.
U.S. Pat. No. 5,814,618 discloses a bidirectional promoter which has multiple tet operator sequences (defined in the specification as enhancers or repressors) and flanking minimal promoters. U.S. Pat. No. 5,955,646 discloses bidirectional heterologous constructs. U.S. Pat. No. 5,368,855 discloses a naturally-occurring bidirectional promoter. U.S. Pat. No. 5,359,142 discloses constructs which have been manipulated to permit variation in enhancement of gene expression. U.S. Pat. No. 5627046 discloses a naturally-occurring bidirectional promoter. U.S. Pat. No. 5,827,693 discloses modified hemoglobin promoters. Significant in this patent is column 12 of the background of the invention, wherein a long list of promoters, some of which are bidirectional, are disclosed. The yeast promoters disclosed are all naturally-occurring ones.
Despite the existence of naturally-occurring bidirectional promoters, it was not clear at the time of the present invention, that any unidirectional promoter can be bidirectionalized. The U.S. Pat. No. 5,814,618 showed that seven repeats of the prokaryotic Tet repressor/operator/inducer sequences, when flanked by two minimal promoters in the presence of tetracyclin inducer, could direct the expression of two genes in eukaryotic cells where raltaed additional prokaryotic proteins/peptides were also expressed. U.S. Pat. No. 5814618 did not teach how to make an eukaryotic polar promoter bidirectional, especially when the specific sequences of the cis elements in an eukaryotic promoter were unknown. In contrast, the current invention describes a novel method by which any eukaryotic polar promoter can be bidirectionalized even at the time when the specific cis elements on the promoter may be not necessarily identified yet. There is ample art to show that stearic hindrance, and other physical as well as chemical barriers makes promoter construction unpredictable.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of these documents.
In the present invention, the inventors have developed a strategy by which a naturally-occurring unidirectional promoter can be bidirectionalized by fusing a minimal promoter at its 5xe2x80x2 end in an opposite orientation to the promoter (FIG. 1), and that the engineered promoter can simultaneously direct the expression of two genes, with one gene on each end.
The present invention comprises artificial nucleic acid constructs comprising a bidirectional promoter having minimal promoter and a common promoter, wherein said minimal promoter is operably linked 5xe2x80x2 to the common promoter, and in opposite orientation to said common promoter. Those artificial nucleic acid constructs, wherein said bidirectional promoter further comprises at least one gene operably linked to said minimal promoters and said common promoter are preferred. In particular, those constructs wherein said minimal promoter is a minimal promoter of a promoter selected from the group consisting of cauliflower mosaic virus 35S RNA promoter (herein 35S), peanut chloritic streak caulimovirus full-length transcript promoter (herein PClSV), the Arabidopsis 12-oxophytodienoic acid-10, 11-reductase gene promoter (herein OPR), and SAG12 are preferred. The common promoter may or may not be defined or known although preferred are artificial nucleic acids comprising common promoter selected from the group consisting of cauliflower mosaic virus 35S RNA promoter (herein 35S), peanut chloritic streak caulimovirus full-length transcript promoter (herein PClSV), the Arabidopsis 12-oxophytodienoic acid-10, 11-reductase gene promoter (herein OPR), and SAG12.
In additional embodiments are artificial nucleic acid constructs which comprise at least one gene and wherein the gene is an agronomically-significant gene. In those embodiments, it is preferred that said agronomically-significant gene is selected from the group consisting of: male sterility; self-incompatibility; foreign organism resistance; improved biosynthetic pathways; environmental tolerance; photosynthetic pathways; and nutrient content and/or fruit ripening; oil biosynthesis; pigment biosynthesis; seed formation; starch metabolism; salt tolerance; cold/frost tolerance; drought tolerance; tolerance to anaerobic conditions; protein content; carbohydrate content (including sugars and starches); amino acid content; and fatty acid content.
Plants comprising the nucleic acid constructs are provided, including, preferably plants selected from the group consisting of: soybean; maize; sugar cane; beet; tobacco; wheat; barley; poppy; rape; sunflower; alfalfa; sorghum; rose; carnation; gerbera; carrot; tomato; lettuce; chicory; pepper; melon; cabbage; oat; rye; cotton; flax; potato; pine; walnut; citrus (including oranges, grapefruit etc.); hemp; oak; rice; petunia; orchids; Arabidopsis; broccoli; cauliflower; brussel sprouts; onion; garlic; leek; squash; pumpkin; celery; pea; bean (including various legumes); strawberries; grapes; apples; pears; peaches; banana; palm; cocoa; cucumber; pineapple; apricot; plum; sugar beet; lawn grasses; maple; triticale; safflower; peanut; and olive.
Plant parts comprising a nucleic acid constructs are also provided, particularly those selected from the group consisting of cells, seeds, embryos, leaves, roots, and stems.
The present invention also provides methods to transfer nucleic acid into a plant cell, comprising contacting a nucleic acid constructs of the present invention to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell. In particular, there are provided methods to impart agronomically-significant characteristics to a plant, comprising contacting a nucleic acid constructs of the present invention to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell.
By xe2x80x9cagronomically-significantxe2x80x9d it is meant any trait of a plant which is recognized by members of the agricultural industry as desirable.
xe2x80x9cCommonxe2x80x9d or xe2x80x9cunidirectionalxe2x80x9d or xe2x80x9cpolarxe2x80x9d promoter means a naturally-occurring unidirectional promoter, or a unidirectional promoter which is an obvious variation of a naturally-occurring unidirectional promoter.
xe2x80x9cOperatively linkedxe2x80x9d the spatial relationship of two or more pieces of DNA such that the referent piece(s) is capable of effecting an intended genetic outcome of another referent sequence. The pieces may be joined together by recombinant DNA techniques, including, but not limited to, restriction enzyme digestion and ligations.
By xe2x80x9cplantxe2x80x9d it is meant one or more plant seed, plant embryo, plant part or whole plant. The plant may be an angiosperm (monocot or dicot), gymnosperm, man-made or naturally-occurring.
By xe2x80x9cproteinsxe2x80x9d it is meant any compounds which comprise amino acids, including peptides, polypeptides, fusion proteins, etc.
Moreover, for the purposes of the present invention, the term xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d entity refers to one or more of that entity; for example, xe2x80x9ca proteinxe2x80x9d or xe2x80x9ca nucleic acid moleculexe2x80x9d refers to one or more of those compounds or at least one compound. As such, the terms xe2x80x9caxe2x80x9d (or xe2x80x9canxe2x80x9d), xe2x80x9cone or morexe2x80x9d and xe2x80x9cat least onexe2x80x9d can be used interchangeably herein. It is also to be noted that the terms xe2x80x9ccomprisingxe2x80x9d, xe2x80x9cincludingxe2x80x9d, and xe2x80x9chavingxe2x80x9d can be used interchangeably. Furthermore, a compound xe2x80x9cselected from the group consisting ofxe2x80x9d refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds.